Exhaled breath from an individual contains thousands of molecules that can provide useful information about the individual's health. Breath analysis therefore has the potential to provide relatively inexpensive, rapid, noninvasive methods for detecting and/or monitoring a variety of metabolic processes and diseases. Breath analysis also provides utility in other applications, such as environmental monitoring, security, etc. (Cikach, F S and Dweik, R A (2012) “Cardiovascular Biomarkers in Exhaled Breath,” Prog. Cardiovasc. Dis. 55(1):34-43).
Exhaled breath contains mostly water vapor, as well as smaller amounts of volatile, semi-volatile, and non-volatile particles derived from the upper and lower portions of the respiratory system (Effros, R M et al. (2005) “Epithelial lining fluid solute concentrations in chronic obstructive lung disease patients and normal subjects,” J. Appl. Physiol. 99:1286-1292; Horvath, I et al. (2005) “Exhaled breath condensate: methodological recommendations and unresolved questions,” Eur. Respir. J. 26:523-548; McKenzie, J H et al. (2012) “Collection of Aerosolized Human Cytokines Using Teflon® Filters,” PLoS ONE, vol. 7, issue 5, page 1-11, e35814). It has been shown that approximately 98% of the particles produced during tidal breathing are under 1 μm (Fairchild, C I and Stampfer, J F (1987) “Particle concentration in exhaled breath,” Am. Ind. Hyg. Assoc. J. 48:948-949; Papineni, R S and Rosenthal, F S (1997) “The size distribution of droplets in the exhaled breath of healthy human subjects,” J. Aerosol Med. 10:105-116; Edwards, D A et al. (2004) “Inhaling to mitigate exhaled bioaerosols,” Proc. Natl. Acad. Sci. USA 101:17383-17388; Morawska, L et al. (2008) “Size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities,” J. Aerosol. Sci. 40:256-269). For example, in a previous study of subjects infected with influenza, it was found that the subjects produced 67 to 8500 particles per liter of air, and that 87% of the particles were under 1 μm (Fabian, P et al. (2008) “Influenza virus inhuman exhaled breath: an observational study,” PLoS ONE 3:e2691).
Numerous volatile organic compounds (VOCs) have been identified in exhaled human breath, some of which have been associated with metabolic pathways and processes (see Cikach, F. S. & Dweik, R. A. (2012), supra, Prog. Cardiovasc. Dis. 55(1):34-43). However, far fewer studies have been conducted on non-volatile compounds in exhaled breath. Non-volatile substances incorporated in aerosolized particles are believed to derive from the respiratory tract lining fluid (RTLF), which is a heterogeneous lining layer that covers the respiratory epithelium (Scheideler, L et al. (1993) “Detection of nonvolatile macromolecules in breath. A possible diagnostic tool?” Am. Rev. Respir. Dis. 148:778). In the upper airways (from the trachea to the approximately the 15th generation of airway divisions), the RTLF includes a gel layer over a sol layer. It contains high levels of mucins, secreted by goblet cells and submucosal mucus gland mucous cells. Lipids, lipid metabolites, proteoglycans, proteases and antimicrobial proteins and peptides are also constituents of the RTLF in the upper airways. Alveoli begin to appear at about the 15th generation of airway divisions and are more frequent until at about the 23rd division the airways terminate in alveolar sacks. The alveolar epithelium is very thin, and coated with surfactant, a complex mixture comprised of glycerophospholipids (˜80%), neutral lipids (˜10%), and proteins (˜10%). (Levitzky, M. G. (2013). Chapter 1. Function and Structure of the Respiratory System. In Pulmonary Physiology, (New York, N.Y.: The McGraw-Hill Companies); Widdicombe, J. (2012). Airway Epithelium. Colloquium Series on Integrated Systems Physiology: From Molecule to Function 4, 1-148).
It has long been thought that during tidal breathing, exhaled aerosol particles (e.g., droplets of RTLF) are generated by shear forces produced by air flow acting on the airway lining fluid, thereby entraining particles composed of mucus, surfactant and pathogens (King, M et al. (1985) “Clearance of mucus by simulated cough,” J. Appl. Physiol. 58:1776-1782; Moriarty, J A and Grotberg, J B (1999) “Flow-induced instabilities of a mucus-serous bilayer,” J. Fluid Mech. 397:1-22; see also Leith, D et al. (1986) “Cough” in M J Macklem (ed). Handbook of Physiology, The Respiratory System, Section 3, Vol. III, Part 1, Bethesda, Md.: American Physiological Society, pp. 315-336). However, more recent evidence has strongly supported the hypothesis that RTLF droplets are produced from the destabilization of the lining fluid during the reopening of collapsed small airways and alveoli during breathing (Edwards, D A et al. (2004), supra., Proc. Natl. Acad. Sci. USA 101:17383-17388; see also Johnson, G R and Morawska, L (2009) “The mechanism of breath aerosol formation,” J. Aerosol Med. Pulm. Drug Deliv. 22:229-237). Identifying the origin of these particles is important when interpreting studies of exhaled breath biomarkers (Shahid, S K et al. (2002) “Increased interleukin-4 and decreased interferon-gamma in exhaled breath condensate of children with asthma,” Am. J. Respir. Crit. Care Med., 165:1290-1293; Garey, K W et al. (2004) “Markers of inflammation in exhaled breath condensate of young healthy smokers,” Chest. 125: 22-26; Rosias, P P et al. (2004) “Childhood asthma: exhaled markers of airway inflammation, asthma control score, and lung function tests,” Pediatr. Pulmonol. 38:107-114; Carpagnano, G E et al. (2002) “Interleukin-6 is increased in breath condensate of patients with non-small cell lung cancer,” Int. J. Biol. Markers, 17:141-145; Leung, T F et al. (2004) “Increased macrophage-derived chemokine in exhaled breath condensate and plasma from children with asthma,” Clin Exp Allergy, 34:786-791; and Rosias, P et al. (2004) “Exhaled breath condensate: a space odessey, where no one has gone before,” Eur. Respir. J. 24:189-190), metals in exhaled breath (Broding, H C et al. (2009) “Comparison between exhaled breath condensate analysis as a marker for cobalt and tungsten exposure and biomonitoring in workers of a hard metal alloy processing plant,” Int. Arch. Occup. Environ. Health. 82:565-573; Goldoni, M et al. (2008) “Chromium in exhaled breath condensate and pulmonary tissue of non-small cell lung cancer patients,” Int. Arch. Occup. Environ. Health, 81:487-493; Mutti, A et al. (2006) “Exhaled metallic elements and serum pneumoproteins in asymptomatic smokers and patients with COPD or asthma,” Chest. 129:1288-1297), pathogens such as viruses (Fabian, P et al. (2008), supra., PLoS ONE 3:e2691; Huynh, K N et al. (2008) “A new method for sampling and detection of exhaled respiratory virus aerosols,” Clin. Infect. Dis. 46:93-95) and bacteria (Fennelly, K P et al. (2004) “Cough-generated aerosols of Mycobacterium tuberculosis: a new method to study infectiousness,” Am. J. Respir. Crit. Care Med. 169:604-609).
Thus, there has been great interest in noninvasive techniques for the collection and analysis of biomarkers present in aerosolized particles. The availability of sampling methods that are convenient for the patient and can be performed on a regular basis would greatly facilitate the early detection of airway disease and the monitoring of disease progression and the patient's response to therapy. Moreover, non-invasive methods are unlikely to harm the airways during sampling.
Conventional techniques for obtaining samples containing biomarkers from exhaled breath have primarily focused on the collection of exhaled breath condensate (EBC). EBC samples include a mixture of three main components (Horvath, I et al. (2005), supra., Eur. Respir. J. 26:523-548). The most abundant component of EBC samples is liquid water (>99%) formed from the condensation of water vapor present in the warm exhaled air, saturated with water vapor as it leaves the respiratory tract. The second and third components of EBC samples are water-soluble volatile and non-volatile droplets that are aerosolized from the RTLF and are present in significantly smaller amounts than the water vapor component (Horvath, I et al. (2005), supra., Eur. Respir. J. 26:523-548; Kietzmann, D et al. (1993) “Hydrogen peroxide in expired breath condensate of patients with acute respiratory failure and with ARDS,” Intensive Care Med. 19:78-81; Effros, R M et al. (2002) “Dilution of respiratory solutes in exhaled condensates,” Am. J. Respir. Crit. Care Med. 165:663-669; Horvath, I et al. (2009) “Exhaled biomarkers in lung cancer,” Eur. Respir. J. 34:261-275; Kazani, S and Israel, E (2010) “Exhaled breath condensates in asthma: diagnostic and therapeutic implications,” J. Breath Res. 4:047001; Loukides, S et al. (2011) “Exhaled breath condensate in asthma: from bench to bedside,” Curr. Med. Chem. 18:1432-1443; McKenzie, J H et al. (2012), supra, PLoS ONE, vol. 7, issue 5, page 1-11, e35814).
Collection of EBC samples is typically accomplished through means whereby a subject breathes tidally into a chilled collection device for a fixed period of time (e.g., 10 minutes). The exhaled breath is then condensed in the device, and as much of the resulting condensate as possible is collected. Unfortunately, the significant amount of liquid water from condensed water vapor present in EBC samples dilutes the inherently low concentrations of certain analytes, particularly non-volatile biomarkers from RTLF droplets, to levels that are at or below the detection threshold of most conventional assays. For example, analyte concentrations may be diluted by 20000-fold or more by the condensed liquid water using conventional EBC collection systems. Moreover, inefficient collection of aerosolized droplets of RTLF results in substantial sample loss. EBC devices collect aerosol particles, including droplets of RTLF, by allowing turbulence to result in impaction on the walls of the device. However, variable airflow rates during exhalation result in variable turbulence and impaction. For example, the aerosol particle collection efficiency of most conventional EBC devices is less than 25%. Some EBC collection methods also provide for efficient impaction of collected EBC into a liquid medium, but, thereby also diluting analyte concentrations (see U.S. Pat. No. 9,617,582).
The inefficient collection of exhaled, RTLF droplets containing non-volatile aerosol particles and the extensive collection of water vapor using conventional EBC collection methods, combined with most assay sensitivity limitations, has therefore created significant problems with reproducibility and validity of biomarker measurements (Horvath, I et al. (2005), supra., Eur. Respir. J. 26:523-548; Kazani, S and Israel, E (2010) “Exhaled breath condensates in asthma: diagnostic and therapeutic implications,” J. Breath Res. 4:047001; Loukides, S et al. (2011), supra., Curr. Med. Chem. 18:1432-1443; Sack, U et al. (2006) “Multiplex analysis of cytokines in exhaled breath condensate,” Cytometry A. 69:169-172; Bayley, D L et al. (2008) “Validation of assays for inflammatory mediators in exhaled breath condensate,” Eur. Respir. J. 31:943-948; Sapey, E et al. (2008) “The validation of assays used to measure biomarkers in exhaled breath condensate,” Eur. Respir. J. 32:1408-1409).
Another approach to collecting particles (e.g. such as RTLF droplets) from exhaled breath provides for the use of a conventional three-stage inertial impactor. Such impactors rely on inertia of the particles within a flow path. In particular, aerosol particles with greater inertia attach to a plate in the first stage, while those with less inertia flow through nozzles and enter into the following stages. Although such methods have been reported to successfully collect some protein from RTLF droplets, the obstacles with recovery from the impaction plate material have inhibited their use. Thus far, successful reports primarily require impaction onto silicon wafers and analysis by mass spectroscopy and are not amenable to other analytical techniques (e.g. immunoassay or PCR).
Accordingly, there is a need for systems and methods for collecting and analyzing exhaled breath aerosol that overcome some or all of the problems associated with conventional systems and methodologies.